Polysaccharide of different molecular weights from Dunaliella salina (polysaccharide of Dunaliella salina,PD) was prepared by 40%, 60% and 80% of ethanol fractional precipitation, which were named PD-40, PD-60 and PD-80, respectively.The physicochemical properties such as HPGPC, FT-IR, monosaccharide composition, SEM, TGA and Congo red assay of the three polysaccharide components were analyzed, and in vitro immune activity was also evaluated in RAW 264.7 cell culture.Results indicated that when the ethanol concentration increased to 40%, 60% and 80% (v/v), three PD components with decreasing molecular weight (PD-40>PD-60>PD-80) were produced sequentially, and there was no difference in structure or thermal stability based on the FT-IR and TGA analysis.Meanwhile, three PD components were all composed of 10 monosaccharide residues with different molar ratios, among them, the PD40 component had the largest content and molecular weight (MW=2.31×107), accounting for 61.4% of the total PD mass, and also had the highest galactose (Gal) molar ratio and uronic acid content.Additionally, PD40 components could significantly enhance the production of NO and ROS as well as the phagocytic activity in RAW 264.7 cells.Ethanol fractional precipitation is an effective method for isolating immunoactive PD from D. salina while reducing solvent consumption.
[1] BOROWITZKA L J, BOROWITZKA M A, MOULTON T P.The mass culture of Dunaliella salina for fine chemicals:From laboratory to pilot plant[J].Hydrobiologia, 1984, 116(1):115-121.
[2] 庄坤.两种工业杜氏盐藻生长的优化和β-胡萝卜素积累机制的研究[D].沈阳:沈阳农业大学, 2020.
ZHUANG K.Growth optimization and β-carotene accumulation in two industrial strains of Dunaliella salina[D].Shenyang:Shenyang Agricultural University, 2020.
[3] BROWN M R.The amino-acid and sugar composition of 16 species of microalgae used in mariculture[J].Journal of Experimental Marine Biology and Ecology, 1991, 145(1):79-99.
[4] FABREGAS J, GARCÍA D, FERNANDEZ-ALONSO M, et al.In vitro inhibition of the replication of haemorrhagic septicaemia virus (VHSV) and African swine fever virus (ASFV) by extracts from marine microalgae[J].Antiviral Research, 1999, 44(1):67-73.
[5] 郑维发, 王莉, 石枫, 等.盐藻水提物对小鼠细胞免疫功能的影响[J].中成药, 2004, 26(12):1031-1036.
ZHENG W F, WANG L, SHI F, et al.Effects on cell immunity in mice by water extract of Dunaliella salina[J].Chinese Traditional Patent Medicine, 2004, 26(12):1031-1036.
[6] CHEN H G, XIAO R X, ZHOU X.Study on the extraction, purification, partial chemical characterization and anti-alcohol liver injury activity of Mori Fructus polysaccharides[J].New Journal of Chemistry, 2020, 44(46):20060-20070.
[7] CHOU C H, SUNG T J, HU Y N, et al.Chemical analysis, moisture-preserving, and antioxidant activities of polysaccharides from Pholiota nameko by fractional precipitation[J].International Journal of Biological Macromolecules, 2019, 131:1021-1031.
[8] GAO W H, ZHANG P L, LIN P Z, et al.Comparison of Litchi polysaccharides extracted by four methods:Composition, structure and in vitro antioxidant activity[J].International Journal of Food Science & Technology, 2020, 55(3):1343-1350.
[9] 徐锡莲. 盐藻胞外多糖的分离与结构研究[D].杭州:浙江大学, 2007.
XU X L.Study on the separation and structure of extracellular polysaccharide from Dunaliella salina[D].Hangzhou:Zhejiang University, 2007.
[10] 刘云鹤, 周鸣谦, 朱蓓蕾.盐藻水溶性多糖的提取和纯化[J].淮海工学院学报(自然科学版), 2012, 21(1):88-91.
LIU Y H, ZHOU M Q, ZHU B L.Extraction and purification of polysaccharides from Duanaliella salina[J].Journal of Huaihai Institute of Technology (Natural Sciences Edition), 2012, 21(1):88-91.
[11] 李秀霞, 孙协军, 王珍, 等.盐藻多糖提取及初步纯化[J].渤海大学学报(自然科学版), 2012, 33(4):334-340;349.
LI X X, SUN X J, WANG Z, et al.Extraction and preliminary purification of polysaccharide from Dunaliella salina[J].Journal of Bohai University (Natural Science Edition), 2012, 33(4):334-340;349.
[12] 戴军. 杜氏盐藻多糖的提取、分离分析及其生物活性的研究[D].无锡:江南大学, 2007.
DAI J.Study on isolation, identification and bioactivity of polysaccharides from Duanaliella salina[D].Wuxi:Jiangnan University, 2007.
[13] 钟闰, 吴思伟, 何秀苗, 等.杜氏盐藻胞外多糖抗肿瘤活性及其机制研究[J].食品工业科技, 2020, 41(22):126-133.
ZHONG R, WU S W, HE X M, et al.Antitumor activity and mechanism of exopolysaccharide from Dunaliella salina[J].Science and Technology of Food Industry, 2020, 41(22):126-133.
[14] DUBOIS M, GILLES K A, HAMILTON J K, et al.Colorimetric method for determination of sugars and related substances[J].Analytical Chemistry, 1956, 28(3):350-356.
[15] BRADFORD M M.A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[J].Analytical Biochemistry, 1976, 72(1-2):248-254.
[16] BITTER T, MUIR H M.A modified uronic acid carbazole reaction[J].Analytical Biochemistry, 1962, 4(4):330-334.
[17] LI L Q, SONG A X, YIN J Y, et al.Anti-inflammation activity of exopolysaccharides produced by a medicinal fungus Cordyceps sinensis Cs-HK1 in cell and animal models[J].International Journal of Biological Macromolecules, 2020, 149:1042-1050.
[18] HU X T, GOFF H D.Fractionation of polysaccharides by gradient non-solvent precipitation:A review[J].Trends in Food Science & Technology, 2018, 81:108-115.
[19] 付海宁, 赵峡, 于广利, 等.盐藻多糖单糖组成分析的四种色谱方法比较[J].中国海洋药物, 2008, 27(4):30-34.
FU H N, ZHAO X, YU G L, et al.Comparison of four chromatographic methods for monosaccharide composition analysis of Dunaliella salina polysaccharide[J].Chinese Journal of Marine Drugs, 2008, 27(4):30-34.
[20] GUO R, AI L Z, CAO N N, et al.Physicochemical properties and structural characterization of a galactomannan from Sophora alopecuroides L.seeds[J].Carbohydrate Polymers, 2016, 140:451-460.
[21] CHEN G J, BAI Y X, ZENG Z Q, et al.Structural characterization and immunostimulatory activity of heteropolysaccharides from fuzhuan brick tea[J].Journal of Agricultural and Food Chemistry, 2021, 69(4):1368-1378.
[22] 唐方媛, 张娅娣, 刘咏, 等.白玉菇多糖理化性质及免疫活性研究[J].合肥工业大学学报(自然科学版), 2024, 47(2):283-288.
TANG F Y, ZHANG Y D, LIU Y, et al.Physicochemical properties and immune activities of polysaccharides from white Hypsizygus marmoreus[J].Journal of Hefei University of Technology (Natural Science), 2024, 47(2):283-288.
[23] 贺妍, 戴阿利, 王怡, 等.北虫草多糖诱导巨噬细胞极化的机制研究[J].延安大学学报(自然科学版), 2024, 43(1):8-12.
HE Y, DAI A L, WANG Y, et al.Study on the mechanism of polarization of macrophages induced by Cordyceps militaris polysaccharides[J].Journal of Yan’an University (Natural Science Edition), 2024, 43(1):8-12.
[24] KOHCHI C, INAGAWA H, NISHIZAWA T, et al.ROS and innate immunity[J].Anticancer Research, 2009, 29(3):817-821.
[25] HUANG Q L, SIU K C, WANG W Q, et al.Fractionation, characterization and antioxidant activity of exopolysaccharides from fermentation broth of a Cordyceps sinensis fungus[J].Process Biochemistry, 2013, 48(2):380-386.
[26] 赵丽, 金周雨, 荆波, 等.多糖受体与巨噬细胞吞噬作用关系研究进展[J].食品科学, 2019, 40(21):311-316.
ZHAO L, JIN Z Y, JING B, et al.Recent progress in the relationship between polysaccharide receptor and macrophage phagocytosis[J].Food Science, 2019, 40(21):311-316.
